Chapter 4

Flow Cytometry and Cell Activation

Sonia Gavasso

Abstract

Flow cytometry is combined with highly specific fluorophore-conjugated antibodies that will only bind tothe activated forms of molecules. The advances in flow cytometry enable to perform quantitative multi-

plexed analysis of single cells within heterogeneous populations stained with specific antibodies for

phenotyping in conjunction with antibodies to phosphorylated, i.e., activated molecules within signalingpathways. By reactivating signaling pathways in vitro it is possible to collect data on the responsive state of

complex cell populations such as immune cells. In this protocol, peripheral blood mononuclear cells

(PBMC) are stimulated with cytokines for the indicated time in a 37�C/CO2 incubator, fixed immediatelywith paraformaldehyde to freeze signaling, permeabilized with methanol, and then stained simultaneously

with an antibody cocktail to signaling molecules within the JAK-STAT pathway and phenotypic markers

for T-cells and B-cells. The protocol shows a basic four-color method which can be expanded to

potentially study any signaling pathway in a defined cell subset.

Key words: Flow cytometry, PBMC, phosphoprotein, immune cells, signaling, phospho-antibodies,activation, multiparameter, phosphorylation.

1. Introduction

Flow cytometry is developing into an exciting tool for the studyof post-translational modifications at the single cell level. Theadvances in hardware and software, in parallel with commer-cially available reagents enable analysis of signaling networks inprimary samples such as peripheral blood mononuclear cells(PBMC). Highly specific fluorophore-conjugated antibodiesare combined with flow cytometry to perform quantitativemultiparameter analysis of single cells within complex cellpopulation (1).

Gennaro De Libero (ed.), T Cell Protocols: Second Edition, vol. 514� 2009 Humana Press, a part of Springer ScienceþBusiness MediaDOI 10.1007/978-1-60327-527-9_4 Springerprotocols.com

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The potential to combine flow cytometry with fluorophore-conjugated antibodies to specific activated molecules within aparticular signaling cascade makes this technology an innovativetool to analyze the responsive state of immune cells. Immune cellscommunicate through signaling molecules such as cytokines inorder to coordinate a response to pathogens. Intracellularly, thesesignals are among others propagated by phosphatases and kinasesto induce an appropriated cell type specific response (2, 3). Bycombining antibodies that only recognize the phosphorylatedforms of molecules and traditionally used markers for immuno-phenotyping with flow cytometry new approaches to phospho-protein analysis have become available. The technique enablesanalysis of intricate signaling networks within complex cell popu-lations without the need to separate the cells of interest first. It ispossible to gather data on the pertinent signaling pathway in thecell population of interest by reactivating particular pathways invivo or in vitro. Flow cytometry has the unique capability toproduce quantitative data of these phosphorylation events at thesingle cell level.

For this technique to work successfully, experience in flowcytometry is recommended. Besides instrument configuration inyour particular cytometer it is also crucial to be familiar withexcitation and emission of particular fluorophores as well as theirchemical properties, i.e., stability. Furthermore, compensation isinevitable when running a multi-color experiment (4). Manyfluorophore-antibody conjugates are being developed and testedspecifically for flow cytometry and often the antibodies are com-mercially available, already conjugated to fluorophores.

To illustrate the method PBMC are being stimulated with apanel of cytokines, fixed, permeabilized, and stained with cell typespecific markers for T-cells (CD3+) and B-cells (CD20+) in con-junction with antibodies to phosphorylated intracellular signalingmolecules of the JAK-STAT signaling cascade central to immunefunction (Janus Kinases, Signal Transducer and Activator of Tran-scription). The protocol is an example of a multiplexed four-coloranalyses within a heterogeneous cell population. For further read-ing see (5–11).

2. Materials

1. Separation of mononuclear cells from whole blood. Forexample BD Vacutainer CPT tubes (see Note 1)

2. PBS, cell culture grade

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3. Serum-free media that supports PBMC such as BioWhittakerX-Vivo 15

4. Falcon tubes (15 ml)

5. Hemocytometer

6. Trypan blue

7. Cryovials (4�C)

8. DMSO, cell culture grade

9. Serum-free cryopreservation media such as ProFreeze-CDMserum-free media, 4�C

10. Freezing container for repeatable cooling rate such as ‘‘Mr.Frosty’’ Freezing container, Nalgene

11. Isopropanol

12. Warm paraformaldehyde (37�C), 16% paraformaldehydeampoules, Electron Microscopy services, store at room tem-perature (RT), keep away from light sources and use within 1week. Caution: Paraformaldehyde is toxic by inhalation andingestion. Handle with care and use appropriate protectivemeasurements (see Note 2)

13. Methanol, 95%. Store at –20�C.

14. Cell culture tested stimuli such as cytokines : INF-�, INF-�,IL-2, IL-10, IL-6 (see Note 3)

15. Staining buffer: PBS with 1% BSA, pH 7.4, filtered 0.2 mm. Itis recommended to make fresh staining buffer (see Note 2)

16. Prepare cytokine or stimuli working solutions (see Note 3)

17. Phospho-antibodies tested for specificity and titrated, such asthe following phospho-antibodies from BD: pSTAT1-Alexa647, pSTAT6-Alexa488, pSTAT3-Alexa488,pSTAT5-Alexa647, CD3-PE, CD20-PerCP-Cy5.5 (seeNote 4)

18. If applicable uncoated hardware such as 6-well plates for invitro cell activation (see Note 3)

19. FACS tubes

20. Calibration and compensation beads. For Calibur cytometeruse BD Calibrite beads for all four colors (see Note 5)

21. Flow cytometer equipped with Argon laser (448 nm) andRed Diode laser (635 nm); four fluorescent detectors: FL1:530–15 nm, FL2: 585–21 nm, FL3: 670 nm, FL4:661–8 nm. We use a bench top FACSCalibur dual-lasercytometer (BD).

22. Analyzing software for flow cytometry data, such as FlowJo(Treestar)

23. Rotator for incubator, optional

Flow Cytometry and Cell Activation 37

3. Method

Collection and handling of PBMC is described in Section 3.1, invitro activation and phosphostaining protocol in Section 3.2, anddata analysis in Section 3.3.

3.1. Handling of PBMC 1. Prepare: Serum-free media (4�C). ProFreeze media (4�C). Icebucket. Sterile PBS. Mr. Frosty filled with isopropanol

2. Collect whole blood in BD Vacutainer CPT tubes (see Note 1)

3. Process within 2 h according to manufacturer instruction

4. Resuspend PBMC in 4�C X-Vivo 15 serum-free media at15.0 � 106 cells/ml.

5. Work on ice

6. Carefully add chilled ProFreeze-CDM media supplementedwith 15% DMSO to equal volume of cell suspension. Thefinal cell concentration will be 7.50 � 106 cells/ml, with7.5% DMSO.

7. Transfer suspension to 4�C cryovials, place them in Mr. Frostyand leave container at –80�C overnight before transferring toliquid nitrogen for long term storage.

3.2. In Vitro Activation

Staining

1. Prepare:

� –20�C : 95% MeOH� 37�C water bath:

16% paraformaldehyde ampouleSerum-free support media X-VivoAppropriate number of 9 ml X-Vivo media in 10 mlFalcon tubes to transfer PBMC from cryotubes

2. Optimize stimulation time and concentration of stimulus foryour experimental conditions (see Note 3)

3. Titrate the antibodies if necessary (see Note 4)

4. Compensation for fluorescent spillover (see Note 5)

5. Thaw cells quickly in 37�C water bath for 1–2 min, drycryovials and wipe with alcohol. Open tube in cell culturehood and transfer to 9 ml warm X-Vivo when the cell suspen-sion is still partly frozen. You need 0.5–1.0 � 106 cells foreach stimulation/staining combination

6. Spin the cells gently at 250–300 g for about 7–10 min

7. Resuspend cells and add enough serum-free support mediafor in vitro activation at a cell concentration of 0.5–1.0 �106

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cells/ml. Add more media to account for pipetting errors. Letthe cells equilibrate for 1–2 h in incubator. (see Note 3)

8. Split cells into Falcon tubes or FACS tubes, one for eachstimulus plus 1 for unstimulated cells

Make sure you have enough cells to split them for staining at0.5–1.0 � 106 cells/ml for every antibody cocktail. Let cellsrest for an additional 30 min before activation (see Note 3)

9. Add cytokines at a concentration of 20 ng/ml and stimulatecells in incubator for 15 min or as optimized in your pre-liminary experimental setup (see Note 3)

10. Stop reaction with warm PFA at a final concentration of 1.6%.Mix gently by pipetting. PFA is toxic and volatile. Takeappropriate precautions

11. Let cells fix at room temperature for 10 min

12. Spin cells at 500–800 g for 7–10 min. Check your steps.Discard supernatant

13. Resuspend cells in small volume of PBS (e.g., 50 ml). This willprevent clumping of cells when MeOH is added. Check cellsunder the microscope

14. Add ice-cold 95% MeOH. Generally you need about 1 mlMeOH to permeabilize 1�106 cells. Mix well by gentlyvortexing. Important: Check for clumping under the micro-scope. Use a 10 ml pipette if clumping persists. Be patient:cells will un-clump if you resuspended them in PBS beforeyou add MeOH

15. Incubate at room temperature for 10 min

16. To help pelleting of cells in MeOH, add about 1–2 ml of PBS

17. Spin cells at 500–800 g for 7–10 min

18. Wash cells with 1–2 ml PBS

19. Resuspend cells in appropriate volume of PBS. You need 50 mlfor each staining plus 5–10% to account for pipetting errors

20. Prepare antibody cocktails A, B (see Note 4)

A: B:

STAT1-Alexa647 10 ml STAT5-Alexa647 10 ml

STAT6-Alexa488 10 ml STAT3-Alexa488 10 ml

CD3-PE 10 ml CD3-PE 10 ml

CD20-PerCP-Cy5.5 10 ml CD20-PerCP-Cy5.5 10 ml

Staining Buffer 10 ml Staining Buffer 10 ml

Flow Cytometry and Cell Activation 39

21. Add appropriate antibody master mix to corresponding FACStube. Work on ice and protect antibodies from light sources

22. Add 50 ml of cell suspension to appropriate tube, vortex briefly

23. Incubate reaction at room temperature in the dark for 30 min

24. Add 2 ml staining buffer for first wash. Spin 300–500 g for5–10 min

25. Resuspend and repeat step 23

26. Resuspend cells in 200–250 ml of Staining Buffer. If cells aretoo diluted you will spend a lot of time on one sample tocollect enough cells on rare cell subtypes

27. Keep cells cool and dark until flow analysis, for example,Styrofoam box with ice and lid.

3.3. Data Acquisition 1. Calibrate cytometer following instruction for your particularmachine, for example, Calibrite beads

2. Optimize instrument settings with your own cell samples (seeNote 5)

3. If possible, set up instrument to collect enough events for theleast abundant cell subtype, the more the better!

Fig. 4.1. Multidimensional analysis of human PBMC stimulated with either IL-6 (green), IL-4 (red) or left untreated (blue).Cells were fixed and permeabilized following protocol 3.2 and stained simultaneously with antibody cocktail A. Toppanels show superimposed dot plots and histograms for T-cells (CD3+), the bottom panels show B-cells (CD20+). Inoverlays the induction of specific phosphorylation events are clearly identifiable. (See Color Plate 1)

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4. Carefully resuspend cells by vortexing. To obtain data on a per-cell basis it is essential to avoid clumps.

3.4. Data Analysis 1. Export data to FlowJo or similar program

2. Gate cells of interest according to cell markers, Fig. 4.1 andColor Plate 1

3. Make overlay histograms of unstimulated versus stimulatedsamples for every cytokine used, Fig. 4.2 and Color Plate 2

Fig. 4.2. PBMC were stimulated with indicated cytokines, fixed and permeabilized according to protocol 3.2.T-cells (CD3+) and B-cells (CD20+) were gated according to markers while monocytes were gated in scatter plot. Openhistograms represent untreated cells, filled histograms stimulated cells. Induction of phosphorylation is clearly identifi-able (filled yellow histograms). (See Color Plate 2)

Flow Cytometry and Cell Activation 41

4. Visualize data in a heat map as median fluorescent intensity(MFI), log2 transformed: log2 (MFI stimulated/MFI unsti-mulated), Fig. 4.3 and Color Plate 3

4. Notes

1. Blood cells need to be collected with a meticulous attention tohandling. Method, time, temperature, and storage can affect cellviability and performance of this assay (12–15). BD CPT –Vacu-tainer tubes offer sterile collection of peripheral blood which isstable at room temperature for up to 24 h. This offers an off-sitecollection possibility. The tubes are available in different sizes.Make sure you have fitting adaptors for swinging bucket rotors.Cells can also be collected with the traditionally used FicollHypaque method. Serum-free support media has been used

Fig. 4.3. Visualization of the data generated by the FACS analysis following protocol 3.2.The columns represent the cell subsets, T-cells, B-cells, monocytes. Each row repre-sents a cytokine stimulation stained with one of the antibody cocktails and subsequentlyanalyzed for the indicated phosphoprotein. The color of each block represents the foldchange (log2) in MFI in the channel corresponding to the analyzed phophorylatedprotein. (See Color Plate 3)

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for both cryopreservation and in vitro activation. We have beentesting signaling in both serum free media and autologous sera.The basic protocol works for sera as well, but signaling needs tobe evaluated for every new assay.

Cryopreseravtion of cells in general is a very delicate step,especially for rare cells. Cell density in cryovials can vary but inour experience it is of interest to work consistently. If cellviability is lower then expected check this step carefully sincenot all cells react the same to cryopreservation. Depending onpopulations under investigation it may be beneficial to includesera for support.

2. Staining Buffer: Non-specific staining can be higher in fixedand permeabilized cells. It is recommended to add extra pro-tein like BSA to the staining buffer. Sodium azide can be addedto inhibit microbial growth. By making fresh staining bufferfor each run one can avoid the use of toxic sodium azide.

Paraformaldehyde solution, methanol free, is an efficientand rapid penetrating fixative that works particularly well inthis assay to denature and thereby preserve the phosphoryla-tion state of proteins within cells for later staining.

Methanol is an efficient agent for the permeabilization ofPBMC. It has been used at different concentration of 70–100%(6) and is particularly suited for nuclear antigens. Saponin is afurther reagent used for permeabilization, typically at concen-trations of 0.1–0.5%. Some antigens are preserved better withsaponin (9).

3. For in vitro stimulation it is possible to use compounds such asPMA or recombinant peptides to activate the signaling path-way of interest. Careful preparation of the intended analysiswill help in the decision process of which and how many stimulito use in a particular assay and which and how many subpopu-lations to study at first. Generally, if ones intention is to study aparticular pathway it is important to choose a compound thatstrongly activates the signaling cascade studied. If one is inter-ested in evaluating the effect of a drug it may be of interest tostudy more than one pathway simultaneously.

In this protocol the phosphorylation of various STAT mole-cules in response to a panel of cytokines is being assessed.Cytokines are usually shipped lyophilized. Reconstitute themaccording to manufacturer instruction and make sure they havebeen cell cultured tested. To avoid repetitive freeze/thawingaliquot the solutions. You may want to test the reconstitutionmedia for signaling inhibition by stimulating one sample withjust reconstitution media.

Check the literature for a starting point on concentration ofstimulus used in cell culture. To find an appropriate stimula-tion time for your experimental conditions titer the stimulating

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agent for your cell concentration and conditions. If you usemultiple stimuli in one run you may want to compromise andfind a stimulation time that activates all the molecules of inter-est sufficiently. In this particular protocol 15 min are appro-priate for all the cytokines. Perform a serial dilution to findoptimal stimulus concentration for your assay. Take specialcare to work consistently. Cell density and stimulation timeaffect the outcome of this assay and need to be monitoredaccordingly, especially if the analysis of the data is of a quanti-tative nature. Working with antibody master mixes whenapplicable will increase the accuracy.

Hardware must be tested since some cell types tend to stickto plastic, especially if they are activated. Use uncoated hard-ware and rotator if necessary. Check that the cells are in sus-pension before any transfer. Cell loss can be quite substantial.We have used Falcon tubes on a rotator or uncoated plates forstimulation Depending on the pathway and cell type studiedyou may want to let the cells rest for up to 2 h. Cells need toequilibrate and longer resting periods can help cells get back toa basal phosphorylation state after cryopreservation. This willfacilitate better activation.

It is advisable to practice the procedure on cell lines, butkeep in mind that cell lines are not primary cells and that theirsignaling can differ significantly

4. The antibody–fluorophore conjugates used in this assay aretested for their phospho-specificity and their suitability forflow cytometry. Importantly, all antibody–fluorophore conju-gates withstand paraformaldehyde fixation and methanol per-meabilization. We have titrated the antibodies for this assayand found that 10 ml cell surface makers were enough to be ableto easily determine the CD3 and CD20 positive populations.Titration of the intracellular phospho-antibodies in unstimu-lated and stimulated samples showed that 10 ml gave a goodsignal to noise ratio. It is recommended to titrate the antibo-dies for every new assay to insure optimal staining for pheno-typic markers and phospho-antigens under specificexperimental conditions. The links provided below are goodstarting points to plan multicolor experiments. Pay attentionto which kind of permeabilization reagent has been testedfor a particular antibody. Commonly it will be MeOH orsaponin.

If the antibody–fluorophore conjugates are not availablecommercially, you will need to run appropriate tests for yourantibody and fluorophore. Antibody specificity must be checkedby western blot. Test different clones to find those that recog-nize single bands. Keep in mind that antibodies to phosphopro-teins are mostly raised against short peptides in denatured form.Use inhibitors or peptide competitors of the signaling cascade to

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demonstrate activation through phosphorylation by the signal-ing molecule tested. For the choice of fluorophore you need toconsider the wavelengths of the lasers and the filters. Use smalland photostable fluorophores especially for intracellular stain-ing, such as the alexa dyes. Choose bright fluorophores for rareantigens. Dyes and antigens are susceptible to fixation andpermeabilization and need to be evaluated for your particularassay. Fortunately, companies are starting to provide informa-tion on suitability of a particular phospho-antibody conjugatewith certain fixation and permeabilization protocols, see links.Fluorophore conjugation protocols are available from the man-ufacturers themselves. For example, see Invitrogen for thehighly stable alexa dyes.http://www.bdbiosciences.com/docs/Validated_Cell_ Surface_Markers.xlshttp://beckmancoulter.com/literature/Bioresearch/ISAC2004_FMalergue.pdf

5. For multicolor compensation you need single stained cells orcompensation beads for each antibody–fluorophore conju-gate. Keep in mind that monocytes and lymphocytes do nothave the same auto-fluorescence. Compensate lymphocyteswithin the lymphocyte gate and monocytes within the mono-cyte gate. Modern cytometer software can do the compensa-tion for you. To perform a proper compensation you need cellsthat are brightly stained and cells that are unstained or slightlystained within the same tube. If, for example, all PBMC areactivated by a certain compound you can add unstimulatedcells to the same tube. Get help from a knowledgeable person ifthis is your first attempt. You can find a comprehensive discus-sion on the following webpage: http://www. drmr.com/compensation/indexDetail.html

For relative fluorescence intensity measurements your cyt-ometer needs to be calibrated every time to account for instru-ment variability. Follow the manufacturer’s instructions forcalibration. For this four-color flow cytometer protocol youneed Calibrite 3 Beads Kit with PerCp-Cy5.5 and CalibriteAPC and software FACSComp. (If you use a different cyt-ometer use the manufacturer’s instruction for calibration andcompensation.). Optimize the instrument settings with yourown cell samples. Adjust forward and side scatter in order toeasily distinguish lymphocytes from monocytes. Adjust com-pensation for fluorescent spill-over using the single stainedtubes. The compensation settings can be stored and used insubsequent assays. We compensated the two alexa dyes in thelymphocyte gate and checked if the compensation worked forthe monocytes, which it did. Set up the cytometer so thatsufficient events will be collected for the least abundant popu-lation. To get distinct peaks you need to collect a sufficient

Flow Cytometry and Cell Activation 45

number of events. The larger the events the more distinct thepeaks will be. In this case, we determined that a minimum of1,000 events for CD20 B-cells was sufficient to provide analyz-able data.

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14. Maecker HT, Rinfret A, D’Souza P, et al.Standardization of cytokine flow cytometryassays. BMC Immunol 2005;6:13.

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